Volume 113, Issue 12, Pages 2762-2767 (December 2017) Single-Molecule Imaging of Wnt3A Protein Diffusion on Living Cell Membranes  Anna Lippert, Agnieszka A. Janeczek, Alexandre Fürstenberg, Aleks Ponjavic, W.E. Moerner, Roel Nusse, Jill A. Helms, Nicholas D. Evans, Steven F. Lee  Biophysical Journal  Volume 113, Issue 12, Pages 2762-2767 (December 2017) DOI: 10.1016/j.bpj.2017.08.060 Copyright © 2017 The Authors Terms and Conditions

Figure 1 (a) ATTO680-maleimide labeling of Wnt3A protein causes moderate decreases in activity. Wnt3A protein was tagged and run on SDS-PAGE gels and imaged under a fluorescence illumination reader. Fluorescent bands were visible at masses of 66.5 kDa, corresponding to a BSA-only control, and at 40 kDa, the known mass of Wnt3A. (b) Presence of protein was confirmed using silver staining. (c) Western blotting confirmed that the labeled band at 40 kDa was Wnt. (d) ATTO680-maleimide tagging caused decreases in activity in luciferase reporter assays (∗p < 0.05). Biophysical Journal 2017 113, 2762-2767DOI: (10.1016/j.bpj.2017.08.060) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Wnt diffusion is slowed by the presence of the Wnt receptor in S2R+ cells. Fluorophore-labeled Wnt protein was imaged on S2 (red) and S2R+ (blue) cells using highly inclined and laminated optical sheet microscopy ((a) shows a white-light image and (b) shows a single-molecule fluorescence image). (c) A tracking algorithm (26) was used to link fluorescent puncta (red circles, starting (dark red) and current position (filled)) in consecutive frames as described in Materials and Methods (scale bars, 1 μm), resulting in tracks with a localization precision of ∼23 nm (Fig. S3). (d and e) Overlay of tracks with white-light images (scale bars, 5 μm). (f) The ensemble diffusion coefficient was determined by fitting a linear function, which considers static and dynamic errors (32; Fig. S4), to the mean-square displacement (MSD) values versus time. Shown are average MSD values for S2 (red) and S2R+ (blue) cells with mean ± SE, as well as the linear corresponding fit. Biophysical Journal 2017 113, 2762-2767DOI: (10.1016/j.bpj.2017.08.060) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Properties of Wnt S2 membrane associations. (a) Wnt3A binds from solution. The starting positions of Wnt tracks (dark red circles) are evenly distributed on the plasma membrane (white dashed line) of S2 cells demonstrating the probability of single-molecule trajectories occurring is spatially independent. (b) Wnt3A does not transfer between cells. Wnt protein tracks were not found to be crossing cell boundaries of adjacent cells (white dashed line) on S2 cells. The red box indicates the analyzed area. (c) Wnt3A diffuses along cellular processes. Single Wnt3A molecules were also found along cellular processes, suggesting free diffusion along these structures. Wnt tracks are shown in gray with some highlighted tracks in red. Scale bars: left, 10 μm; right, 1 μm. Biophysical Journal 2017 113, 2762-2767DOI: (10.1016/j.bpj.2017.08.060) Copyright © 2017 The Authors Terms and Conditions